Lipoprotein associated phospholipase A2, inhibitors thereof and use of the same in diagnosis and therapy

ABSTRACT

The enzyme Lp-PLA2 in purified form, an isolated nucleic acid molecule encoding Lp-PLA2, the use of an inhibitor of the enzyme Lp-PLA2 in therapy and a method of screening compounds to identify those compounds which inhibit the enzyme.

This is a continuation of application Ser. No. 08/387,858, filed Feb. 24, 1995 now U.S. Pat. No. 5,981,252 which is a national stage of PCT/GB94/01374 filed Jun. 24, 1994.

The present invention relates to the use of inhibitors of an enzyme in the therapy, in particular in the treatment of atherosclerosis. The present invention also relates to the isolation and purification of the enzyme, to isolated nucleic acids encoding the enzyme, to recombinant host cells transformed with DNA encoding the enzyme, to the use of the enzyme in diagnosing a patient's susceptibility to atherosclerosis, and to the use of the enzyme in identifying compounds which are potentially useful for the treatment atherosclerosis.

Lipoprotein Associated Phospholipase A₂ (Lp-PLA₂), also previously known in the art as Platelet Activating Factor Acetyl Hydrolase (PAF acetyl hydrolase). During the conversion of LDL to its oxidised form, Lp-PLA₂ is responsible for hydrolysing the sn-2 ester of oxidatively modified phosphatidylcholine to give lyso-phosphatidylcholine and an oxidatively modified fatty acid. Both of these products of Lp-PLA₂ action are potent chemoattractants for circulating monocytes. As such, this enzyme is thought to be responsible for the accumulation of cells loaded with cholesterol ester in the arteries, causing the characteristic ‘fatty streak’ associated with the early stages of atherosclerosis. Inhibition of the Lp-PLA₂ enzyme would therefore be expected to stop the build up of this fatty streak (by inhibition of the formation of lysophosphatidylcholine), and so be useful in the treatment of atherosclerosis. In addition, it is proposed that Lp-PLA₂ plays a direct role in LDL oxidation. This is due to the poly unsaturated fatty acid-derived lipid peroxide products of Lp-PLA₂ action contributing to and enhancing the overall oxidative process. In keeping with this idea, Lp-PLA₂ inhibitors inhibit LDL oxidation. Lp-PLA₂ inhibitors may therefore have a general application in any disorder that involves lipid peroxidation in conjunction with the enzyme activity, for example in addition to conditions such as atherosclerosis and diabetes other conditions such as rheumatoid arthritis, stroke, myocardial infarction, reperfusion injury and acute and chronic inflammation.

The present invention therefore provides in a first aspect an inhibitor of the enzyme lipoprotein associated Lp-PLA₂ for use in therapy, in particular in the treatment of atherosclerosis. Suitable compounds able to inhibit the Lp-PLA₂ enzyme are known in the art and include for example, the following compounds of structure (I):

in which R is C₁₋₆alkylCONR²;

R² is hydrogen or C₁₋₆alkyl;

X is oxygen, sulphur or —O(C)—;

R¹ is C₈₋₂₀alkyl;

Z is N(R³)₂, ^(⊕)N(R³)₃, SR³, ^(⊕)S(R³)₂, in which each group R³ is the same or different and is C₁₋₆ alkyl, OR², C₁₋₄alkanoyl, imidazolyl or N-methylimidazolyl

Suitably R² is hydrogen or C₁₋₆ alkyl; preferably R² is hydrogen.

Suitably X is oxygen, sulphur or —O(CO)—; preferably X is oxygen

Suitably R¹ is C₈₋₂₀alkyl; preferably R¹ is C₁₆₋₁₈ alkyl

Suitably Z is N(R³)₂, ^(⊕)N(R³)₃, SR³, ^(⊕)S(R³)₂, in which each group R³ is the same or different and is C₁₋₆ alkyl, OR², C₁₋₄alkanoyl, imidazolyl or N-methylimidazolyl; preferably Z is SR³ in which R³ is methyl or OR² in which R² is hydrogen

The compounds of structure (I) can be prepared by processes known to those skilled in the art, for example as described in J Chem Soc Chem Comm., 1993, 70-72; J Org Chem, 1983, 48, 1197 and Chem Phys Lipids, 1984,35,29-37 or procedures analogous thereto.

When used in therapy, the compounds of structure (I) are formulated in accordance with standard pharmaceutical practice.

The compounds of structure (I) and their pharmaceutically acceptable salts which are active when given orally can be formulated as liquids, for example syrups, suspensions or emulsions, tablets, capsules and, lozenges.

A liquid formulation will generally consist of a suspension or solution of the compound or pharmaceutically acceptable salt in a suitable liquid carrier(s) for example, ethanol, glycerine, non-aqueous solvent, for example polyethylene glycol, oils, or water with a suspending agent, preservative, flavouring or colouring agent.

A composition in the form of a tablet can be prepared using any suitable pharmaceutical carrier(s) routinely used for preparing solid formulations. Examples of such carriers include magnesium stearate, starch, lactose, sucrose and cellulose.

A composition in the form of a capsule can be prepared using routine encapsulation procedures. For example, pellets containing the active ingredient can be prepared using standard carriers and then filled into a hard gelatin capsule; alternatively, a dispersion or suspension can be prepared using any suitable pharmaceutical carrier(s), for example aqueous gums, celluloses, silicates or oils and the dispersion or suspension then filled into a soft gelatin capsule.

Typical parenteral compositions consist of a solution or suspension of the compound or pharmaceutically acceptable salt in a sterile aqueous carrier or parenterally acceptable oil, for example polyethylene glycol, polyvinyl pyrrolidone, lecithin, arachis oil or sesame oil. Alternatively, the solution can be lyophilised and then reconstituted with a suitable solvent just prior to administration.

A typical suppository formulation comprises a compound of formula (I) or a pharmaceutically acceptable salt thereof which is active when administered in this way, with a binding and/or lubricating agent such as polymeric glycols, gelatins or cocoa butter or other low melting vegetable or synthetic waxes or fats.

Preferably the composition is in unit dose form such as a tablet or capsule.

Each dosage unit for oral administration contains preferably from 1 to 250 mg (and for parenteral administration contains preferably from 0.1 to 25 mg) of a compound of the formula (I) or a pharmaceutically acceptable salt thereof calculated as the free base.

The daily dosage regimen for an adult patient may be, for example, an oral dose of between 1 mg and 500 mg, preferably between 1 and 250 mg, or an intravenous, subcutaneous, or intramuscular dose of between 0.1 mg and 100 mg, preferably between 0.1 mg and 25 mg, of the compound of the formula (I) or a pharmaceutically acceptable salt thereof calculated as the free base, the compound being administered 1 to 4 times per day. Suitably the compounds will be administered for a period of continuous therapy.

The enzyme, lipoprotein associated Lp-PLA₂ has not hitherto been available in isolated purified form. The present invention therefore provides in a further aspect, the enzyme lipoprotein associated Lp-PLA₂ in purified form. By purified form is meant at least 80%, more preferably 90%, still more preferably 95% and most preferably 99% pure with respect to other protein contaminants.

The enzyme Lp-PLA₂ may be characterised by one or more partial peptide sequences selected from SEQ ID NOs:1, 2, 3, 4, 10 and 11 or by the partial peptide sequence comprising residues 271 to 441 or consisting of residues 1 to 441 of SEQ ID NO:9. The enzyme Lp-PLA₂ may further or alternatively characterised by its molecular weight found to be 45 kDa, at least 45 kDa, 45-47 kDa, 46-47 kDa or 45-50 kDa.

The invention also provides fragments of the enzyme having Lp-PLA₂ activity.

The enzyme can be isolated and purified using the methods hereafter described. Once isolated, the protein sequence of the enzyme can be obtained using standard techniques. In identifying said sequence, a number of protein fragments have been identified, each of which comprises part of the whole sequence of the enzyme. These sequences are themselves novel and form a further aspect of the invention.

This invention also provides isolated nucleic acid molecules encoding the enzyme, including mRNAs, DNAs, cDNAs as well as antisense analogs thereof and biologically active and diagnostically or therapeutically useful fragments thereof.

In particular, the invention provides an isolated nucleic acid molecule consisting of bases 1 to 1361 or 38 to 1361 or comprising the sequence corresponding to bases 848 to 1361 of SEQ ID NO: 9.

This invention also provides recombinant vectors, such as cloning and expression plasmids useful as reagents in the recombinant production of the enzyme, as well as recombinant prokaryotic and/or eukaryotic host cells comprising the novel nucleic acid sequence.

This invention also provides nucleic acid probes comprising nucleic acid molecules of sufficient length to specifically hybridize to the novel nucleic acid sequences.

This invention also provides an antisense oligonucleotide having a sequence capable of binding with mRNAs encoding the enzyme so as to prevent the translation of said mRNA.

This invention also provides transgenic non-human animals comprising a nucleic acid molecule encoding the enzyme. Also provided are methods for use of said transgenic animals as models for mutation and SAR (structure/activity relationship) evaluation as well as in drug screens.

This invention further provides a method of screening compounds to identify those compounds which inhibit the enzyme comprising contacting isolated enzyme with a test compound and measuring the rate of turnover of an enzyme substrate as compared with the rate of turnover in the absence of test compound.

“Recombinant” polypeptides refer to polypeptides produced by recombinant DNA techniques; i.e., produced from cells transformed by an exogenous DNA construct encoding the desired polypeptide. “Synthetic” polypeptides are those prepared by chemical synthesis.

A “replicon” is any genetic element (e.g., plasmid, chromosone, virus) that functions as an autonomous unit of DNA replication in vivo; i.e., capable of replication under its own control.

A “vector” is a replicon, such as a plasmid, phage, or cosmid, to which another DNA segment may be attached so as to bring about the replication of the attached segment.

A “double-stranded DNA molecule” refers to the polymeric form of deoxyribonucleotides (bases adenine, guanine, thymine, or cytosine) in a double-stranded helix, both relaxed and supercoiled. This term refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this term includes double-stranded DNA found, inter alia, in linear DNA molecules (e.g., restriction fragments), viruses, plasmids, and chromosomes. In discussing the structure of particular double-stranded DNA molecules, sequences may be described herein according to the normal convention of giving only the sequence in the 5′ to 3′ direction along the sense strand of DNA.

A DNA “coding sequence of” or a “nucleotide sequence encoding” a particular protein, is a DNA sequence which is transcribed and translated into a polypeptide when placed under the control of appropriate regulatory sequences.

A “promoter sequence” is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3′ direction) coding sequence. Within the promoter sequence will be found a transcription initiation site (conveniently defined by mapping with nuclease S1), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase. Eukaryotic promoters will often, but not always, contain “TATA” boxes and “CAT” boxes.

DNA “control sequences” refers collectively to promoter sequences, ribosome binding sites, polyadenylation signals, transcription termination sequences, upstream regulatory domains, enhancers, and the like, which collectively provide for the expression (i.e., the transcription and translation) of a coding sequence in a host cell.

A control sequence “directs the expression” of a coding sequence in a cell when RNA polymerase will bind the promoter sequence and transcribe the coding sequence into mRNA, which is then translated into the polypeptide encoded by the coding sequence.

A “host cell” is a cell which has been transformed or transfected, or is capable of transformation or transfection by an exogenous DNA sequence.

A cell has been “transformed” by exogenous DNA when such exogenous DNA has been introduced inside the cell membrane. Exogenous DNA may or may not be integrated (covalently linked) into chromosomal DNA making up the genome of the cell. In prokaryotes and yeasts, for example, the exogenous DNA may be maintained on an episomal element, such as a plasmid. With respect to eukaryotic cells, a stably transformed or transfected cell is one in which the exogenous DNA has become integrated into the chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones comprised of a population of daughter cell containing the exogenous DNA.

A “clone” is a population of cells derived from a single cell or common ancestor by mitosis. A “cell line” is a clone of a primary cell that is capable of stable growth in vitro for many generations.

Two DNA or polypeptide sequences are “substantially homologous” or “substantially the same” when at least about 85% (preferably at least about 90%, and most preferably at least about 95%) of the nucleotides or amino acids match over a defined length of the molecule and includes allelic variations. As used herein, substantially homologous also refers to sequences showing identity to the specified DNA or polypeptide sequence. DNA sequences that are substantially homologous can be identified in a Southern hybridization experiment under, for example, stringent conditions, as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See, e.g., “Current Protocols in Mol. Biol.” Vol. I & II, Wiley Interscience, Ausbel et al. (ed.) (1992). Protein sequences that are substantially the same can be identified by proteolytic digestion, gel electrophoresis and microsequencing.

The term “functionally equivalent” intends that the amino acid sequence of the subject protein is one that will exhibit enzymatic activity of the same kind as that of Lp-PLA₂.

A “heterologous” region of a DNA construct is an identifiable segment of DNA within or attached to another DNA molecule that is not found in association with the other molecule in nature.

This invention provides an isolated nucleic acid molecule encoding the enzyme Lp-PLA₂. One means for isolating the coding nucleic acid is to probe a human genomic or cDNA library with a natural or artificially designed probe using art recognized procedures (See for example: “Current Protocols in Molecular Biology”, Ausubel, F. M., et al. (eds.) Greene Publishing Assoc. and John Wiley Interscience, New York, 1989,1992); for example using the protein fragment information disclosed herein. The enzyme of this invention may be made by recombinant genetic engineering techniques. The isolated nucleic acids particularly the DNAs can be introduced into expression vectors by operatively linking the DNA to the necessary expression control regions (e.g. regulatory regions) required for gene expression. The vectors can be introduced into the appropriate host cells such as prokaryotic (e.g., bacterial), or eukaryotic (e.g. yeast, insect or mammalian) cells by methods well known in the art (Ausubel et al.,supra). The coding sequences for the desired proteins having been prepared or isolated, can be cloned into a suitable vector or replicon. Numerous cloning vectors are known to those of skill in the art, and the selection of an appropriate cloning vector is a matter of choice. Examples of recombinant DNA vectors for cloning and host cells which they can transform include the bacteriophage λ (E. coli), pBR322 (E. coli), pACYC177 (E. coli), pKT230 (gram-negative bacteria), pGV1106 (gram-negative bacteria), pLAFR1 (gram-negative bacteria), pME290 (non-E. coli gram-negative bacteria), pHV14 (E. coli and Bacillus subtilis), pBD9 (Bacillus), pIJ61 (Streptomyces), pUC6 (Streptomyces), YIp5 (Saccharomyces), a baculovirus insect cell system, YCp19 (Saccharomyces). See, generally, “DNA Cloning”: Vols. I & II, Glover et al. ed. IRL Press Oxford (1985) (1987) and; T. Maniatis et al. (“Molecular Cloning” Cold Spring Harbor Laboratory (1982).

The gene can be placed under the control of a promoter, ribosome binding site (for bacterial expression) and, optionally, an operator (collectively referred to herein as “control” elements), so that the DNA sequence encoding the desired protein is transcribed into RNA in the host cell transformed by a vector containing this expression construction. The coding sequence may or may not contain a signal peptide or leader sequence. The protein sequences of the present invention can be expressed using, for example, the E. coli tac promoter or the protein A gene (spa) promoter and signal sequence. Leader sequences can be removed by the bacterial host in post-translational processing. See, e.g., U.S. Pat. Nos. 4,431,739; 4,425,437; 4,338,397.

In addition to control sequences, it may be desirable to add regulatory sequences which allow for regulation of the expression of the protein sequences relative to the growth of the host cell. Regulatory sequences are known to those of skill in the art, and examples include those which cause the expression of a gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. Other types of regulatory elements may also be present in the vector, for example, enhancer sequences.

An expression vector is constructed so that the particular coding sequence is located in the vector with the appropriate regulatory sequences, the positioning and orientation of the coding sequence with respect to the control sequences being such that the coding sequence is transcribed under the “control” of the control sequences (i.e., RNA polymerase which binds to the DNA molecule at the control sequences transcribes the coding sequence). Modification of the coding sequences may be desirable to achieve this end. For example, in some cases it may be necessary to modify the sequence so that it may be attached to the control sequences with the appropriate orientation; i.e., to maintain the reading frame. The control sequences and other regulatory sequences may be ligated to the coding sequence prior to insertion into a vector, such as the cloning vectors described above. Alternatively, the coding sequence can be cloned directly into an expression vector which already contains the control sequences and an appropriate restriction site. Modification of the coding sequences may also be performed to alter codon usage to suit the chosen host cell, for enhanced expression.

In some cases, it may be desirable to add sequences which cause the secretion of the polypeptide from the host organism, with subsequent cleavage of the secretory signal. It may also be desirable to produce mutants or analogs of the enzyme of interest. Mutants or analogs may be prepared by the deletion of a portion of the sequence encoding the protein, by insertion of a sequence, and/or by substitution of one or more nucleotides within the sequence. Techniques for modifying nucleotide sequences, such as site-directed mutagenesis, are well known to those skilled in the art. See, e.g., T. Maniatis et al., supra; DNA Cloning, Vols. I and II, supra; Nucleic Acid Hybridization, supra.

A number of prokaryotic expression vectors are known in the art. See, e.g., U.S. Pat. Nos. 4,578,355; 4,440,859; 4,436,815; 4,431,740; 4,431,739; 4,428,941; 4,425,437; 4,418,149; 4,411,994; 4,366,246; 4,342,832; see also U.K. Patent Applications GB 2,121,054; GB 2,008,123; GB 2,007,675; and European Patent Application 103,395. Yeast expression vectors are also known in the art. See, e.g., U.S. Pat. Nos. 4,446,235; 4,443,539; 4,430,428; see also European Patent Applications 103,409; 100,561; 96,491. pSV2neo (as described in J. Mol. Appl. Genet. 1:327-341) which uses the SV40 late promoter to drive expression in mammalian cells or pCDNA1neo, a vector derived from pCDNA1(Mol. Cell Biol. 7:4I25-29) which uses the CMV promoter to drive expression. Both these latter two vectors can be employed for transient or stable(using G418 resistance) expression in mammalian cells. Insect cell expression systems, e.g., Drosophila, are also useful, see for example, PCT applications U.S. Ser. No. 89/05155 and U.S. Ser. No. 91/06838 as well as EP application 88/304093.3.

Depending on the expression system and host selected, the enzyme of the present invention may be produced by growing host cells transformed by an expression vector described above under conditions whereby the protein of interest is expressed. The protein is then isolated from the host cells and purified. If the expression system secretes the protein into growth media, the protein can be purified directly from the media. If the protein is not secreted, it is isolated from cell lysates or recovered from the cell membrane fraction. Where the protein is localized to the cell surface, whole cells or isolated membranes can be used as an assayable source of the desired gene product. Protein expressed bacterial hosts such as E. coli may require isolation from inclusion bodies and refolding. The selection of the appropriate growth conditions and recovery methods are within the skill of the art.

The identification of this novel target for the treatment of atherosclerosis, also leads to a novel diagnostic method to diagnose a patient's susceptibility to developing atherosclerotic disease.

The present invention therefore provides in a still further aspect a diagnostic method comprising isolating a sample of blood from the patient, and assaying said sample for Lp-PLA₂ activity. Patients that are susceptible to atherosclerotic disease are expected to have elevated levels of the Lp-PLA₂ enzyme, and hence the levels of Lp-PLA₂ provides an indication of the patient's susceptibility to atherosclerotic disease. Moreover, Lp-PLA₂ is found located predominantly on dense subfraction(s) of LDL which are known to be very atherogenic. Plasma Lp-PLA₂ levels could therefore provide a ready measure of these very atherogenic small dense LDL particles.

It is expected that the presence of the enzyme in the blood sample of the patient can be assayed by analysis of enzyme activity (i.e. by an assay set up against the purified enzyme as standard); or alternatively by assaying protein content of the sample by using polyclonal or monoclonal antibodies prepared against the purified enzyme. Monoclonal (and polyclonal) antibodies raised against the purified enzyme or fragments thereof are themselves novel and form a further aspect of the invention.

DATA AND EXAMPLES

1. Screen for Lp-PLA₂ Inhibition

Enzyme activity was determined by measuring the rate of turnover of the artificial substrate (A) at 37° C. in 50 mM HEPES (N-2-hydroxyethylpiperazine-N′-2 -ethanesulphonic acid) buffer containing 150 mM NaCl, pH 7.4.

Assays were performed in 96 well titre plates.

Lp-PLA₂ was pre-incubated at 37° C. with vehicle or test compound for 10 min in a total volume of 180 μl. The reaction was then initiated by the addition of 20 μl 10× substrate (A) to give a final substrate concentration of 20 μM. The reaction was followed at 405 nm for 20 minutes using a plate reader with automatic mixing. The rate of reaction was measured as the rate of change of absorbance.

RESULTS

Com- pound No XR¹ R Z IC₅₀(μM) 1 O(CH₂)₁₅CH₃ CH₃CONH N⁺(CH₃)₃ 0.8 2 O(CH₂)₁₇CH₃ CH₃CONH N⁺(CH₃)₃ 3.5 3 O(CH₂)₁₇CH₃ CH₃CONH S⁺(CH₃)₂ 1.0 4 O(CH₂)₁₇CH₃ CH₃CONH SCH₃ 0.08 5 O(CH₂)₁₇CH₃ CH₃CONH OH 0.45 6 O(CH₂)₁₇CH₃ CH₃CONH OAc 0.2 7 O(CH₂)₁₇CH₃ CH₃CONH

0.5 8 O(CH₂)₁₇CH₃ CH₃CONH

0.55 9 O(CH₂)₁₇CH₃ CF₃CONH N⁺(CH₃)₃ 2.5

2. Copper Stimulated LDL Oxidation

Copper stimulated oxidation of LDL is routinely measured by following the increase in conjugated diene formation by monitoring the change in absorption at 234 nm. This assay can be used to study inhibitors of oxidative modification of LDL. FIG. 1 demonstrates that Lp-PLA₂ inhibitors are effective inhibitors of LDL oxidation through a prolongation of the lag phase, using compound 4 as an example.

3. Inhibition of Cu²⁺ Stimulated lyso-phosphatidylcholine (lyso-PtdCho) Formation

A 1 ml aliquot of human LDL (0.25 mg protein/ml) was incubated for 15 min at 37° C. with compound or vehicle. 5 μM Cu²⁺ was then added to allow oxidation/lyso-PtdCho formation to occur. The incubation was terminated by the addition of 3.75 ml chloroform/methanol/c HCl (200:400:5, v/v/v). Following the addition of 1.25 ml chloroform and 1.25 ml 0.1M HCl, the mixture was vortexed and centrifuged. The lower phase was carefully removed and the upper phase re-extracted with an equal volume of synthetic lower phase. The extracts were pooled and dried under nitrogen.

Phospholipids were reconstituted into 50 μl chloroform/methanol (2:1 v/v). 10 μl aliquots were spotted on to pre-run silica gel HPTLC plates and then developed in chloroform/methanol 25-30% methylamine (60:20:5 v/v/v). Plates were subsequently sprayed with the fluorescent indicator, 2-p-toluidinylnaphthalene-6 -sulphonic acid (1 mM in 50 mM Tris/HCl, pH 7.4) to identify phospholipid components. Fluorescence was measured at 222 nm using a CAMAG TLC scanner. Lipoprotein lyso-PulCho content was quantified using a standard curve (0.05-0.5 μg) prepared in parallel.

Compound 4 dose independently inhibits LDL lyso-PtdCho accumulation stimulated by copper ions with an IC₅₀ value of −30 μM.

4. Purification of Lipoprotein Associated Lp-PLA₂

Low density lipoprotein (LDL) was obtained by plasma apheresis. The LDL was dialysed against 0.5 M NaCl, 50 mM MES (4-morpholine ethane sulphonic acid), pH=6.0 overnight at 4° C. Solid CHAPS (3-[(-3-cholamidopropyl)dimethylamino]-1-propane sulphonate) was added to 10 mM and the LDL stirred for 30 minutes to effect solubilisation. The solubilised LDL was pumped onto a pre-equilibrated Blue Sepharose 6FF (Pharmacia) column (2.6×20 cm). The column was then washed with 50 mM MES, 10 mM CHAPS, 0.5 M NaCl, pH=6.0 followed by 50 mM Tris, 10 mM CHAPS, 0.5 M NaCl, pH=8.0 until the absorbance (280 nm) of the eluate reached zero. Lp-PLA₂ was eluted using 50 mM Tris, 10 mM CHAPS, 1.5 M NaCl, pH=8.0. The Lp-PLA₂ fraction was then concentrated and dialysed overnight against 50 mM Tris, 10 mM CHAPS, pH=8.0.

The dialysed Lp-PLA₂ was submitted to anion exchange chromatography on a mono Q column (Pharmacia) using 50 mM Tris, 10 mM CHAPS, pH=8.0 with a NaCl gradient from zero to 0.3 M. The Lp-PLA₂ fractions obtained from the mono Q column were applied directly to a Hi Trap Blue cartridge (Pharmacia). The cartridge was washed with 50 mM Tris, 10 mM CHAPS, 0.5 M NaCl, pH=8.0 until the absorbance of the eluate (280 nm) was zero. Lp-PLA₂ was then eluted using 50 mM Tris, 10 mM CHAPS, 1.5 M NaCl, pH=8.0. This gave Lp-PLA₂ which is greater than 95% pure as shown in FIG. 2. This also demonstrates that the native enzyme is extensively glycosylated.

5. Enzyme Sequence

The purity of the final enzyme preparation was verified by five criteria 1) SDS-polyacrylamide gel electrophoresis gave one band for both native and de-glycosylated forms. 2) Reverse phase high pressure liquid chromatography (RP-HPLC) gave a single peak, 3) The intact preparation gave no results by protein sequencing, implying that the protein was N-terminally blocked and free of any contaminants with open N-terminals, 4) By laser desorbtion mass spectometry only one broad peak was observed with de-glycosylated protein, and 5) none of the sections of extended peptide data from sequencing gave any databse matches indicative of contaminating proteins. Three cleavage strategies were used to obtain internal sequence information; trypsin (after de-glycosylation), cyanogen bromide (methionine cleavage) and BNPS-Skatol (tryptophan cleavage). The resulting peptides were separated by RP-HPLC, collected and sequenced. The accumulated sequence data allowed several extended stretches of primary structure of the Lp-PLA2 enzyme to be verified. These are shown below as Peptides 1, 2, 3 and 4 (SEQ ID Nos 1 to 4). When searched against the National Centre for Biotechnological information (NCBI) non-redundant peptide sequence databases no high similarity matches were obtained. Estimation of the molecular weight of pure, de-glycosylated protein by laser desorption mass spectometry give values in the region of 45-47 kDa (separately 45 kDa and 46-47 kDa), indicating that the sequences constitute approximately 15 to 20% of the protein.

6. Gene Sequence

Three expressed sequence tags (EST's) from human cDNA libraries have been found to have extensive alignments with the Peptide Sequences 1 to 3. These EST's are shown below as Nucleotide Sequences 1 to 3 (SEQ ID Nos: 5 to 7) Nucleotide Sequence 1 is a 420 base sequence derived from a human foetal spleen library. Nucleotide Sequence 2 is a 379 base sequence derived from a 12-week human embryo library. Nucleotide Sequence 3 is a 279 base sequence derived from a T-cell lymphoma library. The identities at both the nucleic acid and amino acid level justified an overlapping alignment of the cDNA of all three ESTs, Nucleotide Sequences 3 (bases 1-278), 1 (bases 1-389) (in reverse orientation) and 2 (bases 1-304) with the Peptide Sequences 1, 2 and 3 (partially) (SEQ ID NOS: 1-3). Beyond these limits, the poor resolution of the raw sequence data precludes accurate base calling.

There are two remaining unassigned peptide sections from Peptides 3 and 4, both of which are expected to be present in the complete protein, -Q-Y-I-N-P-A-V-, and W-L-M-G-N-I-L-R-L-L-F-G-S-M-T-T-P-A-N- (SEQ ID Nos: 3 and 4).

7. Isolation of Full-length Lp-PLA₂ cDNA

The full DNA sequence was determined for the clone (HLTA145) from which the Lymphoma EST (SEQ ID No:7) was derived, giving a total of 572 bases; SEQ ID No:8. There is one base difference between this sequence and the EST (between bases 1 to 256 of the EST); at position 27 of HLTA145 there is an A compared with a T in the EST. This would cause a coding change; L in HLTA145 compared with F in the EST. Clone HLTA145 was used as a radiolabelled probe to screen the Lymphoma cDNA library in order to isolate the full-length Lp-PLA₂ clone. The library was prepared in the bacteriophage λ vector, Unizap XR (Stratagene).

Preparation of the Filters for Screening

The library was plated out at a density of 20,000 plaques per 150 mm petri dish onto E. coli XL-1 Blue host cells (ie. 200,000 plaques on 10 dishes). An overnight of XL-1 Blue was prepared in 100 mls LB/0.2% w/v Maltose/10 mM MgSO₄. The cells were pelleted, resuspended in 50 mls 10 mM MgSO₄ and stored on ice. 180 μl of the library bacteriophage stock (23,400 pfu's) were added to 7 mls XL-1 Blue cells, mixed and divided into 10 aliquots of 615 μl. The 10 tubes were incubated at 37° C. for 30 minutes. 7 mls of molten (@45° C.) top agarose (0.7% w/v agarose in LB) were added, mixed well and poured onto 150 mm LB agar plates (1.5% w/v agar in LB). The plates were inverted and incubated at 37° C. for approximately 7.5 hours. The plates were held at 4° C. until needed.

The plaques were transfered to 132 mm Hybond-N nylon filters (Amersham International) by laying the filters on the plates for 2 minutes (4 minutes for the duplicate). The DNA's on the filters were denatured for 2 minutes (0.5M NaCl, 1.5M NaOH), neutralised for 4 minutes (1.5M NaCl,1.0M Tris pH7.4) and the filters placed on 2×SSC for 1 minute. The filters were then dried and the DNA cross-linked to the filter using a Stratalinker UV 2400 (Stratagene) at 120,000 μJoules/cm².

The filters were pre-hybridised in 1 mM EDTA, 0.5M NaHPO₄, 7% SDS (Church, G M. and Gilbert, W. (1984) PNAS USA 81 p1991-1995) in a Techne HB2 hybridisation oven at 55° C. for 3 hours. Each bottle contained 2 filters and 25 mls prehybridization solution.

Preparation of the Radiolabelled Probe

The probe cDNA (from HLTA 145) was excised from pBluescript II SK+/− as an approximately 600 bp EcoRI-XhoI fragment and approximately 100 ng of gel purified fragment were labelled using 1.2MBq ³²P dATP and 1.2MBq ³²P dCTP by PCR labelling using Taq DNA polymerase (Boehringer Mannheim) and primers designed to prime at the 5′ and 3′ ends of the EST sequence. The labelling reaction was carried out in a total volume of 200 μl and included unlabelled dNTP's at the following concentrations:—

dATP 20 μm

dCTP 20 μm

dGTP 200 μm

dTTP 200 μm

The PCR reaction was carried out over 35 cycles of:—

94° C. for 30s

60° C. for 30s

72° C. for 30s

Screening

The radiolabelled probe was denatured at 98° C. for 5 minutes and divided into 10 aliquots of 20 μl. One aliquot was added per hybridisation bottle. Hybridisation was carried out over 16 hours at 55° C. The filters were washed at 60° C. (1×10 minutes) with 0.1% w/v SDS, 0.1×SSC (50 mls per wash per bottle). The filters were autoradiographed and the films (Fuji Medical X-Ray Film) developed after 5 days exposure.

Duplicate positives were progressed to a secondary screen. The plaques were cored out into 1 ml SM (100 mM NaCl, 10 mM MgSO₄, 1M Tris, pH7.4), titrated and plated onto 90 mm petri dishes at between 20 and 200 pfu's per dish. The secondary screen was carried out as described for the primary screen except the filters were washed at 65° C. The autoradiographs were developed after 16 hours exposure.

DNA Sequencing

The duplicated positive clones from the secondary screen were excised from the λ Unizap XR bacteriophage vector into the Bluescript phagemid (according to the Stratagene manual) for characterisation. One of the clones, carrying an insert of approximately 1.5 kb, was sequenced on both strands (using the USB Sequenase 2.0 DNA sequencing kit) by primer walking (SEQ ID No:9). The cDNA has an open reading frame with the potential to code for a polypeptide of 441 amino acids.

The 3′ region of the full-length cDNA aligns with the HLTA145 sequence with the exception of 3 mismatches (see below). The predicted polypeptide sequence of the lymphoma Lp-PLA₂ is shown as SEQ ID No:9.

Inspection of the full length cDNA (SEQ ID No:9) reveals probable errors in Peptide 3. One of these errors is in the assignment of continuity between V-M which is incompatible with the perfect sequence match with the cDNA after this position. It seems likely that a short peptide, containing the sequence -Q-Y-I-N-P-, co-purified with the longer cyanogen bromide partial cleavage peptide and, by being present in greater quantity, was assigned as the major sequence and contiguous with the subsequent amino acids. The remaining section of Peptide 3 (SEQ ID No:3) and the whole of Peptide 4 (SEQ ID No:4) can be identified in the predicted full length enzyme sequence (SEQ ID No:9). It thus seems likely that Peptide 3 (SEQ ID No:3) is in fact two separate Peptides 5 (SEQ ID No:10) and 6 (SEQ ID No:11). The second probable error has occurred in the transcription from the raw data fro Peptide 3 (SEQ ID No:3) which on checking was consistent with Peptide 5 (SEQ ID No:10) having the sequence -Q-Y-I-N-P-V-A, rather than Q-Y-I-N-P-A-V-.

The 3 base differences are as follows:

1) T is 859 is A in HLTA145; aminoacid change F in full-length, L in HLTA145. (Note that the original EST is identical with the full-length cDNA at position 859).

2) C at 1173 is T in HLTA145; aminoacid change A in full-length, V in HLTA145.

3) T at 1203 is C in HLTA145; aminoacid change L in full-length, S in HLTA145.

The peptide data and the Foetal Spleen EST sequence (SEQ ID No:5) support the full-length cDNA sequence for differences (2) and (3) although the Human Embryo EST (SEQ ID No:6) is identical to the Lymphoma EST (SEQ ID No:7) at position 1173. The Human Embryo EST (SEQ ID No:6) has a further difference (4) corresponding to position 1245 in the full-length Lymphoma sequence (SEQ ID No:9)(comparison between bases 2 to 304 of the Human Embryo EST and the full-length Lymphoma cDNA).

4) A at 1245 is T in the Embryo EST (SEQ ID No:6)(amino acid change D to V in the Embryo EST). Peptide data covering this region supports the Lymphoma DNA sequence (SEQ ID No:9).

The Lp-PLA₂ DNA sequence from 848 to 1361 of SEQ ID No:9 (amino acid residues 271 to 441 of SEQ ID No:9) is the region for which all major data sets agree substantially, ie. the peptide data, the Foetal spleen, full-length Lymphoma and it includes the known active site and is therefore believed to be a significant characterising region for the Lp-PLA₂ enzyme.

The predicted MW for the full reading frame is 50090. This is in excess of that determined for the de-glycosylated, purified protein but post-translational events could account for this discrepancy. The most likely of these are the removal of an N-terminal signal peptide and/or limited proteolytic degradation of the protein C-terminal. The latter could occur in-vivo, during purification, or under the conditions of de-glycosylation.

Diagnostic Method

A sample of blood is taken from a patient, the plasma/serum sample prepared and passed through a dextran sulphate column pre-equilibrated with 0.9% (w\v) NaCl solution. Following washes with the same salt solution Lp-PLA₂ is eluted with a 4.1% (w\v) NaCl solution. Heparin agarose columns can also be used with the wash and elution solutions containing 0.7% and 2.9% NaCl, respectively. Enzyme present in the sample is determined by assaying for either

(a) enzyme activity:

The substrate (A) (see structure in 1) is used to assay Lp-PLA₂ activity by monitoring the absorbance change at 400 nm. Purified enzyme is pre-incubated at 37° C. and substrate (50 μM) is added after 5 minutes. The absorbance change at 400 nm is monitored for 20 minutes. This substrate has previously been reported as a substrate for classical calcium-dependent PLA₂s. (Washburn, W. N. and Dennis, E. A., J.Amer Chem.Soc., 1990, 112, 2040-2041); or

(b) protein content

Total protein content (i.e. enzyme content) can be determined using polyclonal antiserum raised against purified human Lp-PLA₂. The antisera recognises both native and glycosylated enzyme as measured by immunoprecipitation of activity and Western Blot analysis.

Polyclonal antiserum was prepared as follows. Immunisation of rabbits involved mixing 0.5 ml of purified human Lp-PLA₂ (=100 μg) with an equal volume of Freund's complete adjuvant. The final emulsion was given subcutaneously in 4×0.25 ml injections. Boosting using a Freund's incomplete adjuvant\antigen mixture (4×0.25 ml subcut.; dosage=50 μg) took place 4 weeks later. Adequate titre was evident at between 6-8 weeks from initial injection.

IN THE FIGURES

FIG. 1 is a graph of absorbance at 234 nm against time (min) in a study of inhibition of copper (5 μM)—stimulated LDL (150 μg/ml) oxidation by compound 4 vs control vehicle.

FIG. 2 is an analysis the purified Lp-PLA₂ material of Example 4 following separation by polyacrylamide gel electrophoresis. Lanes 2, 4 and 6 respectively after N-deglycosylation.

SEQUENCE DATA

SEQ ID No: 1 -Peptide 1 -M-L-K-L-K-G-D-I-D-S-N-A-A-I-D-L-S-N-K-A-S-L-A-F-L-Q-K-H-L-G-L-H-K-D-F-D-Q- SEQ ID No: 2 -Peptide 2 -W-M-F-P-L-G-D-E-V-Y-S-R-I-P-Q-P-L-F-F-I-N-S-E-Y-F-Q-Y-P-A-N- SEQ ID No: 3 -Peptide 3 -Q-Y-I-N-P-A-V-M-I-T-I-R-G-S-V-H-Q-N-F-A-D-F-T-F-A-T-G- SEQ ID No: 4 -Peptide 4 -W-L-M-G-N-I-L-R-L-L-F-G-S-M-T-T-P-A-N SEQ. ID. No: 5 - Nucleotide Sequence 1 1 AAAAAACCTA TTTTAATCCT AATTGTATTT CTCTATTCCT GAAGAGTTCT 51 GTAACATGAT GTGTTGATTG GTTGTGTTAA TGTTGGTCCC TGGAATAAGA 101 TTCTCATCAT CTCCTTCAAT CAAGCAGTCC CACTGATCAA AATCTTTATG 151 AAGTCCTAAA TGCTTTTGTA AGAATGCTAA TGAAGCTTTG TTGCTAAGAT 201 CAATAGCTGC ATTTGAATCT ATGTCTCCCT TTAATTTGAG CATGTGTCCA 251 ATTATTTTGC CAGTNGCAAA AGTGAAGTCA GCAAAATTCT GGTGGACTGA 301 ACCCCTGATT GTAATCATCT TTCTTTCTTT ATCAGGTGAG TAGCATTTTT 351 TCATTTTTAT GATATTAGCA GGATATTGGA AATATTCAGN GTTGNTAAAA 401 AGNGGNGGCT GAGGGATTCT SEQ. ID. No: 6 - Nucleotide Sequence 2 1 TGCTAATATC ATAAAAATGA AAAAATGCTA CTCACCTGAT AAAGAAAGAA 51 AGATGATTAC AATCAGGGGT TCAGTCCACC AGANTTTTGC TGACTTCACT 101 TTTGCAACTG GCAAAATAAT TGGACACATG CTCAAATTAA AGGGAGACAT 151 AGATTCAAAT GTAGCTATTG ATCTTAGCAA CAAAGCTTCA TTAGCATTCT 201 TACAAAAGCA TTTAGGACTT CATAAAGATT TTGTTCAGTG GGACTGCTTG 251 ATTGAAGGAG ATGATGAGAA TCTTATTCCA GGGACCAACA TTAACACAAC 301 CAATTCAACA CATCATGTTT ACAGAACTTC TTCCAGGGAA TAGGAGGAAA 351 TACAATTGGG GTTTAAAATA GGTTTTTTT SEQ. ID. No: 7 - Nucleotide Sequence 3 1 GAAGAATGCA TTAGATTTAA AGTTTGATAT GGAACAACTG AAGGACTCTA 51 TTGATAGGGA AAAAATAGCA GTAATTGGAC ATTCTTTTGG TGGAGCAACG 101 GTTATTCAGA CTCTTAGTGA AGATCAGAGA TTCAGATGTG GTATTGCCCT 151 GGATGCATGG ATGTTTCCAC TGGGTGATGA AGTATATTCC AGAATTCCTC 201 AGCCCCTCTT TTTTATCAAC TCTGAATATT TCCAATATCC TGCTAATATC 251 ATAAAANTGG AAAAATGCTA CTCACCTGG SEQ. ID. No: 8 - DNA sequence of HLTA145         10         20         30         40         50 AAAATAGCAG TAATTGGACA TTCTTTAGGT GGAGCAACGG TTATTCAGAC         60         70         80         90        100 TCTTAGTGAA GATCAGAGAT TCAGATGTGG TATTGCCCTG GATGCATGGA        110        120        130        140        150 TGTTTCCACT GGGTGATGAA GTATATTCCA GAATTCCTCA GCCCCTCTTT        160        170        180        190        200 TTTATCAACT CTGAATATTT CCAATATCCT GCTAATATCA TAAAAATGAA        210        220        230        240        250 AAAATGCTAC TCACCTGATA AAGAAAGAAA GATGATTACA ATCAGGGGTT        260        270        280        290        300 CAGTCCACCA GAATTTTGCT GACTTCACTT TTGCAACTGG CAAAATAATT        310        320        330        340        350 GGACACATGC TCAAATTAAA GGGAGACATA GATTCAAATG TAGCTATTGA        360        370        380        390        400 TCTTAGCAAC AAAGCTTCAT CAGCATTCTT ACAAAAGCAT TTAGGACTTC        410        420        430        440        450 ATAAAGATTT TGATCAGTGG GACTGCTTGA TTGAAGGAGA TGATGAGAAT        460        470        480        490        500 CTTATTCCAG GGACCAACAT TAACACAACC AATCAACACA TCATGTTACA        510        520        530        540        550 CAACTCTTCA GGAATAGAGA AATACAATTA GGATTAAAAT AGGTTTTTTA        560        570 AAAAAAAAAA AAAAAAAACT CG SEQ. ID. No: 9 - cDNA Sequence of Lymphoma Lp-PLA₂         10        20        30        40        50 TGAGAGACTAAGCTGAAACTGCTGCTCAGCTCCCAAGATGGTGCCACCCA         60        70        80        90       100 AATTGCATGTGCTTTTCTGCCTCTGCGGCTGCCTGGCTGTGGTTTATCCT   L  H  V  L  F  C  L  C  G  C    L  A  V  V  Y  P        110       120       130       140        150 TTTGACTGGCAATACATAAATCCTGTTGCCCATATGAAATCATCAGCATG F  D  W  Q  Y  I  N  P  V  A  H  M  K  S  S  A  W        160       170       180       190       200 GGTCAACAAAATACAAGTACTGATGGCTGCTGCAAGCTTTGGCCAAACTA  V  N  K  I  Q  V  L  M  A  A  A  S  F  G  Q  T  K        210       220       230       240       250 AAATCCCCCGGGGAAATGGGCCTTATTCCGTTGGTTGTACAGACTTAATG   I  P  R  G  N  G  P  Y  S  V  G  C  T  D  L  M        260       270       280       290       300 TTTGATCACACTAATAAGGGCACCTTCTTGCGTTTATATTATCCATCCCA F  D  H  T  N  K  G  T  F  L  R  L  Y  Y  P  S  Q        310       320       330       340       350 AGATAATGATCGCCTTGACACCCTTTGGATCCCAAATAAAGAATATTTTT  D  N  D  R  L  D  T  L  W  I  P  N  K  E  Y  F  W        360       370       380       390       400 GGGGTCTTAGCAAATTTCTTGGAACACACTGGCTTATGGGCAACATTTTG   G  L  S  K  F  L  G  T  H  W  L  M  G  N  I  L        410       420       430       440       450 AGGTTACTCTTTGGTTCAATGACAACTCCTGCAAACTGGAATTCCCCTCT R  L  L  F  G  S  M  T  T  P  A  N  W  N  S  P  L        460       470       480       490       500 GAGGCCTGGTGAAAAATATCCACTTGTTGTTTTTTCTCATGGTCTTGGGG  R  P  G  E  K  Y  P  L  V  V  F  S  H  G  L  G  A        510       520       530       540       550 CATTCAGGACACTTTATTCTGCTATTGGCATTGACCTGGCATCTCATGGG   F  R  T  L  Y  S  A  I  G  I  D  L  A  S  H  G        560       570       580       590       600 TTTATAGTTGCTGCTGTAGAACACAGAGATAGATCTGCATCTGCAACTTA F  I  V  A  A  V  E  H  R  D  R  S  A  S  A  T  Y        610       620       630       640       650 CTATTTCAAGGACCAATCTGCTGCAGAAATAGGGGACAAGTCTTGGCTCT  Y  F  K  D  Q  S  A  A  E  I  G  D  K  S  W  L  Y        660       670       680       690       700 ACCTTAGAACCCTGAAACAAGAGGAGGAGACACATATACGAAATGAGCAG   L  R  T  L  K  Q  E  E  E  T  H  I  R  N  E  Q        710       720       730       740       750 GTACGGCAAAGAGCAAAAGAATGTTCCCAAGCTCTCAGTCTGATTCTTGA V  R  Q  R  A  K  E  C  S  Q  A  L  S  L  I  L  D        760       770       780       790       800 CATTGATCATGGAAAGCCAGTGAAGAATGCATTAGATTTAAAGTTTGATA  I  D  H  G  K  P  V  K  N  A  L  D  L  K  F  D  M        810       820       830       840       850 TGGAACAACTGAAGGACTCTATTGATAGGGAAAAAATAGCAGTAATTGGA   E  Q  L  K  D  S  I  D  R  E  K  I  A  V  I  G        860       870       880       890       900 CATTCTTTTGGTGGAGCAACGGTTATTCAGACTCTTAGTGAAGATCAGAG H  S  F  G  G  A  T  V  I  Q  T  L  S  E  D  Q  R        910       920       930       940       950 ATTCAGATGTGGTATTGCCCTGGATGCATGGATGTTTCCACTGGGTGATG  F  R  C  G  I  A  L  D  A  W  M  F  P  L  G  D  E        960       970       980       990      1000 AAGTATATTCCAGAATTCCTCAGCCCCTCTTTTTTATCAACTCTGAATAT   V  Y  S  R  I  P  Q  P  L  F  F  I  N  S  E  Y       1010      1020      1030      1040      1050 TTCCAATATCCTGCTAATATCATAAAAATGAAAAAATGCTACTCACCTGA F  Q  Y  P  A  N  I  I  K  M  K  K  C  Y  S  P  D       1060      1070      1080      1090      1100 TAAAGAAAGAAAGATGATTACAATCAGGGGTTCAGTCCACCAGAATTTTG  K  E  R  K  M  I  T  I  R  G  S  V  H  Q  N  F  A       1110      1120      1130      1140      1150 CTGACTTCACTTTTGCAACTGGCAAAATAATTGGACACATGCTCAAATTA   D  F  T  F  A  T  G  K  I  I  G  H  M  L  K  L       1160      1170      1180      1190      1200 AAGGGAGACATAGATTCAAATGCAGCTATTGATCTTAGCAACAAAGCTTC K  G  D  I  D  S  N  A  A  I  D  L  S  N  K  A  S       1210      1220      1230      1240      1250 ATTAGCATTCTTACAAAAGCATTTAGGACTTCATAAAGATTTTGATCAGT L  A  F  L  Q  K  H  L  G  L  H  K  D  F  D  Q  W       1260      1270      1280      1290      1300 GGGACTGCTTGATTGAAGGAGATGATGAGAATCTTATTCCAGGGACCAAC   D  C  L  I  E  G  D  D  E  N  L  I  P  G  T  N       1310      1320      1330      1340      1350 ATTAACACAACCAATCAACACATCATGTTACAGAACTCTTCAGGAATAGA I  N  T  T  N  Q  H  I  M  L  Q  N  S  S  G  I  E       1360 GAAATACAATT  K  Y  N  . SEQ. ID. No: 10 -Peptide 5 -Q-Y-I-N-P-V-A- SEQ. ID. No: 11 -Peptide 6 -M-I-T-I-R-G-S-V-H-Q-N-F-A-D-F-T-F-A-T-G-

                   #             SEQUENCE LISTING (1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 11 (2) INFORMATION FOR SEQ ID NO: 1:      (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 37 amino  #acids           (B) TYPE: amino acid           (C) STRANDEDNESS:           (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: peptide    (iii) HYPOTHETICAL: NO     (iv) FRAGMENT TYPE:      (v) FRAGMENT TYPE: internal     (vi) ORIGINAL SOURCE:      (x) FEATURE:     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #1: Met Leu Lys Leu Lys Gly Asp Ile Asp Ser As #n Ala Ala Ile Asp Leu 1               5    #                10   #                15 Ser Asn Lys Ala Ser Leu Ala Phe Leu Gln Ly #s His Leu Gly Leu His             20       #            25       #            30 Lys Asp Phe Asp Gln         35 (2) INFORMATION FOR SEQ ID NO: 2:      (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 30 amino  #acids           (B) TYPE: amino acid           (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: peptide    (iii) HYPOTHETICAL: NO      (v) FRAGMENT TYPE: internal     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #2: Trp Met Phe Pro Leu Gly Asp Glu Val Tyr Se #r Arg Ile Pro Gln Pro 1               5    #                10   #                15 Leu Phe Phe Ile Asn Ser Glu Tyr Phe Gln Ty #r Pro Ala Asn             20       #            25       #            30 (2) INFORMATION FOR SEQ ID NO: 3:      (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 27 amino  #acids           (B) TYPE: amino acid           (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: peptide    (iii) HYPOTHETICAL: NO      (v) FRAGMENT TYPE: internal     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #3: Gln Tyr Ile Asn Pro Ala Val Met Ile Thr Il #e Arg Gly Ser Val His 1               5    #                10   #                15 Gln Asn Phe Ala Asp Phe Thr Phe Ala Thr Gl #y             20       #            25 (2) INFORMATION FOR SEQ ID NO: 4:      (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 19 amino  #acids           (B) TYPE: amino acid           (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: peptide    (iii) HYPOTHETICAL: NO      (v) FRAGMENT TYPE: internal     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #4: Trp Leu Met Gly Asn Ile Leu Arg Leu Leu Ph #e Gly Ser Met Thr Thr 1               5    #                10   #                15 Pro Ala Asn (2) INFORMATION FOR SEQ ID NO: 5:      (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 420 base  #pairs           (B) TYPE: nucleic acid           (C) STRANDEDNESS: double           (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: cDNA    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #5: AAAAAACCTA TTTTAATCCT AATTGTATTT CTCTATTCCT GAAGAGTTCT GT #AACATGAT     60 GTGTTGATTG GTTGTGTTAA TGTTGGTCCC TGGAATAAGA TTCTCATCAT CT #CCTTCAAT    120 CAAGCAGTCC CACTGATCAA AATCTTTATG AAGTCCTAAA TGCTTTTGTA AG #AATGCTAA    180 TGAAGCTTTG TTGCTAAGAT CAATAGCTGC ATTTGAATCT ATGTCTCCCT TT #AATTTGAG    240 CATGTGTCCA ATTATTTTGC CAGTNGCAAA AGTGAAGTCA GCAAAATTCT GG #TGGACTGA    300 ACCCCTGATT GTAATCATCT TTCTTTCTTT ATCAGGTGAG TAGCATTTTT TC #ATTTTTAT    360 GATATTAGCA GGATATTGGA AATATTCAGN GTTGNTAAAA AGNGGNGGCT GA #GGGATTCT    420 (2) INFORMATION FOR SEQ ID NO: 6:      (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 379 base  #pairs           (B) TYPE: nucleic acid           (C) STRANDEDNESS: double           (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: cDNA    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #6: TGCTAATATC ATAAAAATGA AAAAATGCTA CTCACCTGAT AAAGAAAGAA AG #ATGATTAC     60 AATCAGGGGT TCAGTCCACC AGANTTTTGC TGACTTCACT TTTGCAACTG GC #AAAATAAT    120 TGGACACATG CTCAAATTAA AGGGAGACAT AGATTCAAAT GTAGCTATTG AT #CTTAGCAA    180 CAAAGCTTCA TTAGCATTCT TACAAAAGCA TTTAGGACTT CATAAAGATT TT #GTTCAGTG    240 GGACTGCTTG ATTGAAGGAG ATGATGAGAA TCTTATTCCA GGGACCAACA TT #AACACAAC    300 CAATTCAACA CATCATGTTT ACAGAACTTC TTCCAGGGAA TAGGAGGAAA TA #CAATTGGG    360 GTTTAAAATA GGTTTTTTT              #                   #                   #379 (2) INFORMATION FOR SEQ ID NO: 7:      (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 279 base  #pairs           (B) TYPE: nucleic acid           (C) STRANDEDNESS: double           (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: cDNA    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #7: GAAGAATGCA TTAGATTTAA AGTTTGATAT GGAACAACTG AAGGACTCTA TT #GATAGGGA     60 AAAAATAGCA GTAATTGGAC ATTCTTTTGG TGGAGCAACG GTTATTCAGA CT #CTTAGTGA    120 AGATCAGAGA TTCAGATGTG GTATTGCCCT GGATGCATGG ATGTTTCCAC TG #GGTGATGA    180 AGTATATTCC AGAATTCCTC AGCCCCTCTT TTTTATCAAC TCTGAATATT TC #CAATATCC    240 TGCTAATATC ATAAAANTGG AAAAATGCTA CTCACCTGG       #                   #   279 (2) INFORMATION FOR SEQ ID NO: 8:      (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 572 base  #pairs           (B) TYPE: nucleic acid           (C) STRANDEDNESS: double           (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: cDNA    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #8: AAAATAGCAG TAATTGGACA TTCTTTAGGT GGAGCAACGG TTATTCAGAC TC #TTAGTGAA     60 GATCAGAGAT TCAGATGTGG TATTGCCCTG GATGCATGGA TGTTTCCACT GG #GTGATGAA    120 GTATATTCCA GAATTCCTCA GCCCCTCTTT TTTATCAACT CTGAATATTT CC #AATATCCT    180 GCTAATATCA TAAAAATGAA AAAATGCTAC TCACCTGATA AAGAAAGAAA GA #TGATTACA    240 ATCAGGGGTT CAGTCCACCA GAATTTTGCT GACTTCACTT TTGCAACTGG CA #AAATAATT    300 GGACACATGC TCAAATTAAA GGGAGACATA GATTCAAATG TAGCTATTGA TC #TTAGCAAC    360 AAAGCTTCAT CAGCATTCTT ACAAAAGCAT TTAGGACTTC ATAAAGATTT TG #ATCAGTGG    420 GACTGCTTGA TTGAAGGAGA TGATGAGAAT CTTATTCCAG GGACCAACAT TA #ACACAACC    480 AATCAACACA TCATGTTACA GAACTCTTCA GGAATAGAGA AATACAATTA GG #ATTAAAAT    540 AGGTTTTTTA AAAAAAAAAA AAAAAAAACT CG        #                   #         572 (2) INFORMATION FOR SEQ ID NO: 9:      (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 1361 base  #pairs           (B) TYPE: nucleic acid           (C) STRANDEDNESS: double           (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: cDNA    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO     (ix) FEATURE:           (A) NAME/KEY: CDS           (B) LOCATION: 38..1360     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #9: TGAGAGACTA AGCTGAAACT GCTGCTCAGC TCCCAAG ATG GTG CCA  #CCC AAA TTG      55                    #                   #     Met Val Pro Pro Lys Leu                    #                   #       1            #    5 CAT GTG CTT TTC TGC CTC TGC GGC TGC CTG GC #T GTG GTT TAT CCT TTT      103 His Val Leu Phe Cys Leu Cys Gly Cys Leu Al #a Val Val Tyr Pro Phe              10      #             15      #             20 GAC TGG CAA TAC ATA AAT CCT GTT GCC CAT AT #G AAA TCA TCA GCA TGG      151 Asp Trp Gln Tyr Ile Asn Pro Val Ala His Me #t Lys Ser Ser Ala Trp          25          #         30          #         35 GTC AAC AAA ATA CAA GTA CTG ATG GCT GCT GC #A AGC TTT GGC CAA ACT      199 Val Asn Lys Ile Gln Val Leu Met Ala Ala Al #a Ser Phe Gly Gln Thr      40              #     45              #     50 AAA ATC CCC CGG GGA AAT GGG CCT TAT TCC GT #T GGT TGT ACA GAC TTA      247 Lys Ile Pro Arg Gly Asn Gly Pro Tyr Ser Va #l Gly Cys Thr Asp Leu  55                  # 60                  # 65                  # 70 ATG TTT GAT CAC ACT AAT AAG GGC ACC TTC TT #G CGT TTA TAT TAT CCA      295 Met Phe Asp His Thr Asn Lys Gly Thr Phe Le #u Arg Leu Tyr Tyr Pro                  75  #                 80  #                 85 TCC CAA GAT AAT GAT CGC CTT GAC ACC CTT TG #G ATC CCA AAT AAA GAA      343 Ser Gln Asp Asn Asp Arg Leu Asp Thr Leu Tr #p Ile Pro Asn Lys Glu              90      #             95      #            100 TAT TTT TGG GGT CTT AGC AAA TTT CTT GGA AC #A CAC TGG CTT ATG GGC      391 Tyr Phe Trp Gly Leu Ser Lys Phe Leu Gly Th #r His Trp Leu Met Gly         105           #       110           #       115 AAC ATT TTG AGG TTA CTC TTT GGT TCA ATG AC #A ACT CCT GCA AAC TGG      439 Asn Ile Leu Arg Leu Leu Phe Gly Ser Met Th #r Thr Pro Ala Asn Trp     120               #   125               #   130 AAT TCC CCT CTG AGG CCT GGT GAA AAA TAT CC #A CTT GTT GTT TTT TCT      487 Asn Ser Pro Leu Arg Pro Gly Glu Lys Tyr Pr #o Leu Val Val Phe Ser 135                 1 #40                 1 #45                 1 #50 CAT GGT CTT GGG GCA TTC AGG ACA CTT TAT TC #T GCT ATT GGC ATT GAC      535 His Gly Leu Gly Ala Phe Arg Thr Leu Tyr Se #r Ala Ile Gly Ile Asp                 155   #               160   #               165 CTG GCA TCT CAT GGG TTT ATA GTT GCT GCT GT #A GAA CAC AGA GAT AGA      583 Leu Ala Ser His Gly Phe Ile Val Ala Ala Va #l Glu His Arg Asp Arg             170       #           175       #           180 TCT GCA TCT GCA ACT TAC TAT TTC AAG GAC CA #A TCT GCT GCA GAA ATA      631 Ser Ala Ser Ala Thr Tyr Tyr Phe Lys Asp Gl #n Ser Ala Ala Glu Ile         185           #       190           #       195 GGG GAC AAG TCT TGG CTC TAC CTT AGA ACC CT #G AAA CAA GAG GAG GAG      679 Gly Asp Lys Ser Trp Leu Tyr Leu Arg Thr Le #u Lys Gln Glu Glu Glu     200               #   205               #   210 ACA CAT ATA CGA AAT GAG CAG GTA CGG CAA AG #A GCA AAA GAA TGT TCC      727 Thr His Ile Arg Asn Glu Gln Val Arg Gln Ar #g Ala Lys Glu Cys Ser 215                 2 #20                 2 #25                 2 #30 CAA GCT CTC AGT CTG ATT CTT GAC ATT GAT CA #T GGA AAG CCA GTG AAG      775 Gln Ala Leu Ser Leu Ile Leu Asp Ile Asp Hi #s Gly Lys Pro Val Lys                 235   #               240   #               245 AAT GCA TTA GAT TTA AAG TTT GAT ATG GAA CA #A CTG AAG GAC TCT ATT      823 Asn Ala Leu Asp Leu Lys Phe Asp Met Glu Gl #n Leu Lys Asp Ser Ile             250       #           255       #           260 GAT AGG GAA AAA ATA GCA GTA ATT GGA CAT TC #T TTT GGT GGA GCA ACG      871 Asp Arg Glu Lys Ile Ala Val Ile Gly His Se #r Phe Gly Gly Ala Thr         265           #       270           #       275 GTT ATT CAG ACT CTT AGT GAA GAT CAG AGA TT #C AGA TGT GGT ATT GCC      919 Val Ile Gln Thr Leu Ser Glu Asp Gln Arg Ph #e Arg Cys Gly Ile Ala     280               #   285               #   290 CTG GAT GCA TGG ATG TTT CCA CTG GGT GAT GA #A GTA TAT TCC AGA ATT      967 Leu Asp Ala Trp Met Phe Pro Leu Gly Asp Gl #u Val Tyr Ser Arg Ile 295                 3 #00                 3 #05                 3 #10 CCT CAG CCC CTC TTT TTT ATC AAC TCT GAA TA #T TTC CAA TAT CCT GCT     1015 Pro Gln Pro Leu Phe Phe Ile Asn Ser Glu Ty #r Phe Gln Tyr Pro Ala                 315   #               320   #               325 AAT ATC ATA AAA ATG AAA AAA TGC TAC TCA CC #T GAT AAA GAA AGA AAG     1063 Asn Ile Ile Lys Met Lys Lys Cys Tyr Ser Pr #o Asp Lys Glu Arg Lys             330       #           335       #           340 ATG ATT ACA ATC AGG GGT TCA GTC CAC CAG AA #T TTT GCT GAC TTC ACT     1111 Met Ile Thr Ile Arg Gly Ser Val His Gln As #n Phe Ala Asp Phe Thr         345           #       350           #       355 TTT GCA ACT GGC AAA ATA ATT GGA CAC ATG CT #C AAA TTA AAG GGA GAC     1159 Phe Ala Thr Gly Lys Ile Ile Gly His Met Le #u Lys Leu Lys Gly Asp     360               #   365               #   370 ATA GAT TCA AAT GCA GCT ATT GAT CTT AGC AA #C AAA GCT TCA TTA GCA     1207 Ile Asp Ser Asn Ala Ala Ile Asp Leu Ser As #n Lys Ala Ser Leu Ala 375                 3 #80                 3 #85                 3 #90 TTC TTA CAA AAG CAT TTA GGA CTT CAT AAA GA #T TTT GAT CAG TGG GAC     1255 Phe Leu Gln Lys His Leu Gly Leu His Lys As #p Phe Asp Gln Trp Asp                 395   #               400   #               405 TGC TTG ATT GAA GGA GAT GAT GAG AAT CTT AT #T CCA GGG ACC AAC ATT     1303 Cys Leu Ile Glu Gly Asp Asp Glu Asn Leu Il #e Pro Gly Thr Asn Ile             410       #           415       #           420 AAC ACA ACC AAT CAA CAC ATC ATG TTA CAG AA #C TCT TCA GGA ATA GAG     1351 Asn Thr Thr Asn Gln His Ile Met Leu Gln As #n Ser Ser Gly Ile Glu         425           #       430           #       435 AAA TAC AAT T              #                   #                   #      1361 Lys Tyr Asn     440 (2) INFORMATION FOR SEQ ID NO: 10:      (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 7 amino  #acids           (B) TYPE: amino acid           (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: peptide    (iii) HYPOTHETICAL: NO      (v) FRAGMENT TYPE: internal     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #10: Gln Tyr Ile Asn Pro Val Ala 1               5 (2) INFORMATION FOR SEQ ID NO: 11:      (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 20 amino  #acids           (B) TYPE: amino acid           (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: peptide    (iii) HYPOTHETICAL: NO      (v) FRAGMENT TYPE: internal     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #11: Met Ile Thr Ile Arg Gly Ser Val His Gln As #n Phe Ala Asp Phe Thr 1               5    #                10   #                15 Phe Ala Thr Gly             20 

What is claimed is:
 1. A method of diagnosis of a patient's susceptibility to atherosclerosis which comprises taking a sample of blood from the patient and analyzing said sample for the presence of the enzyme lipoprotein phospholipase A₂ (Lp-PLA₂) using polyclonal or monoclonal antibodies raised against the enzyme, wherein elevated levels of Lp-PLA₂ in said sample are indicative of the patient being susceptible to atherosclerosis. 